Coagulase-negative staphylococci (CoNS), notably Staphylococcus epidermidis, are a predominant cause of hospital-acquired infections and treatment can be difficult owing to biofilm formation and antibiotic resistance. This ability to form a multi-layered biofilm depends, in part, on the production of the surface polysaccharide termed poly-N-acetyl-glucosamine (PNAG). Previous work has demonstrated that PNAG is an in vivo expressed antigen and the target for protective immunity for both S. epidermidis and Staphylococcus aureus. However, production of PNAG varies widely among clinical isolates of CoNS, potentially limiting the use for this surface polymer as the sole component of a vaccine against staphylococcal strains. Therefore, additional potential vaccine candidates need to be investigated. S. epidermidis and other related CoNS elaborate, as extracellular polymers, poly-?-DL-glutamic acid (DL-PGA) to facilitate growth and survival in the human host. These polymers are also produced by Bacillus spp., including B. licheniformis and more notably B. anthracis, and antibody to the PGA mediates protective immunity to anthrax. Preliminary work has shown that mouse antibodies raised to a B. licheniformis DL-PGA-conjugate vaccine can mediate killing of various S. epidermidis strains by human polymorphonuclear leukocytes and complement in an in vitro opsonophagocytic assay. Moreover, the opsonophagocytic killing was independent of PNAG production, as both PNAG positive and negative clinical isolates were similarly opsonized and killed. The major hypothesis being evaluated is that an optimized vaccine that would target S. epidermidis should be multi-component and include PNAG and D- and/or L-glutamic acid in combination in order to provide maximum coverage against staphylococcal clinical isolates and/or obtain a synergistic effect from the individual components. Multiple vaccines candidates will be synthesized including high molecular weight D- and L-PGA, 20-mer synthetic PGA peptides of either the D or L configuration and dPNAG conjugated to the carrier protein tetanus toxoid. The in vitro opsonophagocytic assay will be used as the first screen for potentially protective antibodies. Highly opsonic antibodies and vaccines that elicit them will be further tested for their immunoprotective efficacy by passive and active immunization against challenge with multiple strains of CoNS in a murine catheter-infection model. Insights gained by these studies will help us to evaluate immunotherapeutic strategies for control and prevention of infection by CoNS. Project Narrative: Over the past 2 decades, Staphylococcus epidermidis has become one of the most prevalent pathogens involved in hospital-acquired infections. Importantly many strains of S. epidermidis are resistant to commonly used antibiotics, underscoring the need to find new approaches to prevention and treatment of infection such as identifying novel molecular targets for vaccine development. This application proposes to investigate the vaccine potential of two different S. epidermidis surface polymers termed poly-DL-glutamic acid and deacetylated poly-N-acetyl-glucosamine (dPNAG) either alone or in combination as components of a novel vaccine against this pathogen.